ABSTRACT: BACKGROUND:Male sterility has tremendous scientific and economic importance in hybrid seed production. Identification and characterization of a stable male sterility gene will be highly beneficial for making hybrid seed production economically feasible. In soybean, eleven male-sterile, female-fertile mutant lines (ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, msMOS, and msp) have been identified and mapped onto various soybean chromosomes, however the causal genes responsible for male sterility are not isolated. The objective of this study was to identify and functionally characterize the gene responsible for the male sterility in the ms4 mutant. RESULTS:The ms4 locus was fine mapped to a 216 kb region, which contains 23 protein-coding genes including Glyma.02G243200, an ortholog of Arabidopsis MALE MEIOCYTE DEATH 1 (MMD1), which is a Plant Homeodomain (PHD) protein involved in male fertility. Isolation and sequencing of Glyma.02G243200 from the ms4 mutant line showed a single base insertion in the 3rd exon causing a premature stop codon resulting in truncated protein production. Phylogenetic analysis showed presence of a homolog protein (MS4_homolog) encoded by the Glyma.14G212300 gene. Both proteins were clustered within legume-specific clade of the phylogenetic tree and were likely the result of segmental duplication during the paleoploidization events in soybean. The comparative expression analysis of Ms4 and Ms4_homologs across the soybean developmental and reproductive stages showed significantly higher expression of Ms4 in early flowering (flower bud differentiation) stage than its homolog. The functional complementation of Arabidopsis mmd1 mutant with the soybean Ms4 gene produced normal stamens, successful tetrad formation, fertile pollens and viable seeds, whereas the Ms4_homolog was not able to restore male fertility. CONCLUSIONS:Overall, this is the first report, where map based cloning approach was employed to isolate and characterize a gene responsible for the male-sterile phenotype in soybean. Characterization of male sterility genes may facilitate the establishment of a stable male sterility system, highly desired for the viability of hybrid seed production in soybean. Additionally, translational genomics and genome editing technologies can be utilized to generate new male-sterile lines in other plant species.
